Rhesus D antigen (also referred to in the art as RhD antigen, Rhesus factor, and/or Rh factor) is an antigen which may be present on the surface of human red blood cells. Those individuals whose red blood cells have this antigen are usually referred to as “RhD-positive”, while those individuals whose red blood cells do not have this antigen are referred to as “RhD-negative”.
A person who is RhD-negative and has never been exposed to the RhD antigen will not produce anti-RhD antibodies (antibodies against the RhD antigen). However, transfer of RhD-positive blood to a RhD-negative individual will lead to sensitisation (immunization) of the RhD-negative individual against the RhD antigen. This can lead to a number of complications. In particular, where a RhD-negative woman gives birth to a RhD-positive infant there is a risk of small amounts of the infant's blood entering the maternal circulation, causing the mother to produce anti-RhD antibodies. Whilst this will not normally harm the first baby, should the now immunized mother fall pregnant with another RhD positive child then maternal anti-RhD antibodies may cross the placenta and attack the infant's blood cells, leading to a condition known as haemolytic disease of the newborn (HDN).
Anti-RhD antibodies are therefore routinely administered to RhD-negative patients where there is a risk of exposure to RhD-positive blood, in order to prevent the patient from becoming immunized against the RhD-positive blood. For example, a RhD-negative patient may be given anti-RhD antibodies: prior to and/or shortly after giving birth to or having an abortion of an RhD-positive baby; after any incident during pregnancy which may have lead to bleeding across the placenta; as a routine preventative measure during pregnancy; or prior to or soon after any transfusion of blood components containing RhD-positive red blood cells.
Traditionally, the anti-RhD antibodies used have been polyclonal antibodies obtained from the blood plasma of RhD negative volunteers who have been repeatedly immunized against RhD-positive red blood cells. However, the use of polyclonal antibodies has a number of recognised drawbacks, not least of which are the continuing need for a number of volunteer donors sufficient to meet the demand for antibody, and the risk of contamination of the antibody preparation with any viruses or other pathogens that may be present in the donor's blood.
Whereas polyclonal antibodies constitute antibodies secreted by a number of different plasma cells, and thus constitute a mixture of immunoglobulin molecules secreted against a specific antigen and potentially recognising a variety of epitopes, monoclonal antibodies are produced from cells that are all clones of a single parent cell, and thus constitute a homogeneous population of antibodies, as is well known in the art. The cell lines from which monoclonal antibodies are produced are developed and cultured in-vitro, and this means monoclonal antibodies have the potential to be produced as and when required both in large amounts and at high levels of purity. Accordingly, monoclonal anti-RhD antibodies have a number of potential advantages over the polyclonal anti-RhD antibody preparations that have traditionally been used.
A number of techniques for producing human monoclonal antibodies in general, and human monoclonal anti-RhD antibodies in particular, have been described. For example, EP-A2-0251440 discloses an anti-RhD monoclonal antibody producing heterohybridoma formed by fusion of non-Ig secreting mouse mylenoma cells with an anti-RhD Ig producing population of Epstein Barr virus (EBV) transformed human lymphocytes.
U.S. Pat. No. 5,665,356 describes the production of human monoclonal anti-RhD antibodies having certain defined characteristics, produced by culturing selected EBV-transformed human B-lymphocytes.
U.S. Pat. No. 6,312,690 describes the production anti-RhD monoclonal antibodies by recombinant techniques. An EBV immortalized human cell line producing an anti-Rhesus D monoclonal antibody called D7C2 was selected. The sequences encoding the variable regions of the heavy (H) and light (L) chains of D7C2 were cloned, sequenced, and inserted into a recombinant baculovirus expression vector under the control of a strong baculovirus promoter. Insect cells transfected with the recombinant baculovirus were cultured, and the recombinant D7C2 monoclonal antibody recovered from the cell supernatant.
US-A1-2003/0175969 describes a method for preparing a anti-RhD monoclonal antibodies capable of activating effector cells expressing FcγRIII, comprising: a) purifying monoclonal antibodies obtained from cell lines selected from human B lymphocyte heterohybridomas, or recombinant animal or human cell lines (such as CHO-K, CHO-Lec10, CHO Lec-1, CHO Pro-5, CHO dhfr-, Wil-2, Jurkat, Vero, Molt-4, COS-7, HEK293, YB2/0, BHK, K6H6, NSO, SP2/0-Ag 14 and P3X63Ag8.653 cells); b) adding each antibody obtained in step a) to a different reaction mixture comprising RhD-positive red blood cells, effector cells comprising cells expressing FcγRIII, polyvalent IgGs; and c) determining the percentage lysis of the target cells and selecting the monoclonal antibodies which activate the effector cells causing significant lysis of the RhD-positive red blood cells.
U.S. Pat. No. 6,475,787 discloses a method for preparing monoclonal antibodies, in which a suitable eukaryotic host cell is transformed with a DNA sequence encoding an antibody heavy chain and a DNA sequence encoding an antibody light chain, the two sequences being linked to different amplifiable marker genes so as to allow differential amplification of the heavy and light chain DNAs in order to optimize the relative gene copy numbers of the heavy and light chain DNAs. In a preferred embodiment the host cell is a Chinese Hamster Ovary (CHO) cell which is DHFR deficient (i.e. incapable of producing dihydrofolate reductase), one of the amplifiable marker genes is an adenosine deaminase (ADA) gene, and the other is a DHFR gene. Amplification of the DNA encoding one antibody chain and linked in the ADA gene can then be achieved by treating the recombinant cells with increasing concentrations of 2′-deoxycoformycin, whilst amplification of the DNA encoding the other antibody chain and linked in the DHFR gene is achieved by treating the cell with increasing concentrations of methotrexate (MTX).
Nevertheless, there remains a need for further anti-RhD monoclonal antibodies and methods for the production thereof.